Project Summary The human genome exists in the cell nucleus as chromatin, a complex of the DNA with histone proteins. Though genetic information is encoded in the DNA sequence, another layer of information, is encoded in the histone proteins, specifically in the form of post-translational modifications (PTMs). This layer of information is often referred to as epigenetics, and provides instructions on how the genome is to be regulated. Chromatin and the epigenetic content, is highly dynamic, constantly restructuring in response to developmental and environmental cues. One of the most important questions in biology is how this information is interpreted by transcriptional and other regulatory complexes, leading to gene regulation and cell fate. Histone modifications are ?read? through small subdomains within the regulatory complexes called reader domains, and specificity for a unique modification state is thought to be achieved through the integrated activity of multiples of these domains. However, though much is known about the association of reader domains with fragments of histones, the molecular mechanims underlying how they associate with histones in a chromatin relevent context, or how they function together to readout a specific histone modification state, are not well understood. This research program addresses this fundamental question in chromatin regulation. We are pioneering the use of NMR spectroscopy to study the association of reader domains with the basic unit of chromatin, the nucleosome. We are combining this with X-ray crystallography, fluorescence spectroscopy, biolayer interferometry, and basic biochemistry for an overall multidisciplinary approach to building models of these complexes. Over the next five years we will determine the thermodynamic and structural basis of association of reader domains from the BAF chromatin remodeling and Polycomb histone modifying complexes with nucleosomes, the influence of adjacent chromatin binding domains, and the functional consequence of these interactions. Long-term, we will build towards an understand of how multiple reader domains in these complexes integrate to allow these regulatory complexes to navigate and respond to a dynamic chromatin substrate. The results of these studies will reveal fundamental mechanisms of chromatin regulation, provide insight into the etiology of a number of human diseases, and lay the groundwork for the development of targeted therapeutics.